Studies on the reductively triggered release of heterocyclic and steroid drugs from 5-nitrothien-2-ylmethyl prodrugs

Article abstract of DOI:10.1016/S0040-4020(03)00481-2

Hypoxia (inadequate concentrations of dioxygen in tissues) is present in several disease states, including cancer and rheumatoid arthritis. Prodrug systems, which after bioreduction, selectively release active drugs in these tissues may be important in therapy. The 5-nitrothien-2-ylmethyl ester of aspirin was synthesised by treatment of 5-nitrothiophene-2-methanol with 2-acetoxybenzoyl chloride, whereas that of prednisolone hemisuccinate was prepared by reaction of prednisolone with 5-nitrothien-2-ylmethyl pentafluorophenyl butanedioate. In chemical model systems, both of these ester-linked potential prodrugs suffered hydrolysis, rather than reductively triggered release of the corresponding drug. 1-(5-Nitrothien-2-ylmethoxy)isoquinolines released the corresponding isoquinolin-1-ones (potent inhibitors of poly(ADP-ribose)polymerase) rapidly upon reduction of the nitro group with sodium borohydride/palladium, showing that these may be useful as reductively triggered prodrugs. In approaches to N-linked potential prodrugs, isoquinolin-1-one and nifedipine (a 1,4-dihydropyridine Ca²⁺ channel antagonist) were alkylated at nitrogen by 2-chloromethylthiophene but the corresponding 5-nitrothien-2-ylmethyl derivatives were synthetically inaccessible.

Full text of DOI:10.1016/S0040-4020(03)00481-2

Tetrahedron 59 (2003) 3437–3444
Studies on the reductively triggered release of heterocyclic
and steroid drugs from 5-nitrothien-2-ylmethyl prodrugs
Sandra Ferrer,a Declan P. Naughtonb and Michael D. Threadgillb
,
*
aDepartment of Medical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK
bDepartment of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
Received 30 January 2003; accepted 21 March 2003
Abstract—Hypoxia (inadequate concentrations of dioxygen in tissues) is present in several disease states, including cancer and rheumatoid
arthritis. Prodrug systems, which after bioreduction, selectively release active drugs in these tissues may be important in therapy. The
5-nitrothien-2-ylmethyl ester of aspirin was synthesised by treatment of 5-nitrothiophene-2-methanol with 2-acetoxybenzoyl chloride,
whereas that of prednisolone hemisuccinate was prepared by reaction of prednisolone with 5-nitrothien-2-ylmethyl pentafluorophenyl
butanedioate. In chemical model systems, both of these ester-linked potential prodrugs suffered hydrolysis, rather than reductively triggered
release of the corresponding drug. 1-(5-Nitrothien-2-ylmethoxy)isoquinolines released the corresponding isoquinolin-1-ones (potent
inhibitors of poly(ADP-ribose)polymerase) rapidly upon reduction of the nitro group with sodium borohydride/palladium, showing that
these may be useful as reductively triggered prodrugs. In approaches to N-linked potential prodrugs, isoquinolin-1-one and nifedipine
(a 1,4-dihydropyridine Ca2þ channel antagonist) were alkylated at nitrogen by 2-chloromethylthiophene but the corresponding 5-nitrothien-
2-ylmethyl derivatives were synthetically inaccessible. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
tissue by design of biologically inactive prodrug systems
which, upon selective bioreduction in hypoxic tissue, would
release known therapeutic drugs only in that tissue. This
would improve greatly the selectivity of biodistribution
of such agents. Denny has described9 such prodrugs as
comprising Trigger, Linker and Effector units. Pre-
viously,10–12 we have reported potential general reductively
activated prodrug systems with redox-sensitive Triggers for
delivery of isoquinolinones, amines and diols. Others have
investigated indolequinones in this way.13,14 The proposed
mechanism for reductively triggered drug release from the
nitroheterocyclylmethyl prodrugs is shown in Scheme 1.
In this mechanism, reduction of the nitro group in the
nitrofurans 1a or nitroimidazoles 1b to the corresponding
In many solid tumours, the vasculature is poorly developed
and structured and, in most tumours, there are regions with
acute or chronic hypoxia.1,2 In these hypoxic tissues, viable
cells are relatively resistant to radiotherapy and to many
chemotherapeutic strategies.1,2 Chronic hypoxia is also
present in rheumatoid arthritis and other disease states.3,4
Prodrugs for selective delivery of drugs to tumours have
attracted much interest, with particular effort being
expended on development of bioreductively activated
cytotoxins.5–8 It is only relatively recently that attention
has been focussed on exploiting the physiological difference
in concentration of O2 between normal and hypoxic tumour
Scheme 1. Proposed mechanism of reported reductively triggered release of drugs from nitrofuranylmethyl prodrugs 1a and from nitroimidazolylmethyl
prodrugs 1b.
Keywords: chronic hypoxia; prodrug; isoquinolin-l-one; nitrothiophene.
*
Corresponding author. Tel.: þ44-1225-386840; fax: þ44-1225-386114; e-mail: m.d.threadgill@bath.ac.uk
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00481-2

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S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
type 1c may be useful for reductively triggered release of
drugs in therapy of cancer, rheumatoid arthritis and other
diseases involving hypoxia.
Figure 1. General structures of O-linked and N-linked nitrothienylmethyl
prodrugs in this study.
2. Results and discussion
amino- or hydroxylamino-heterocycles 2a,b triggers expul-
sion of the drug, owing to the availability of a pair of
electrons, as shown. Using chemical model systems for
the reduction, we have achieved rapid expulsion of ‘drugs’
from O-linked prodrugs, such as carbamates10 and diols,11
and from the N-linked prodrugs 2-(5-nitrofuran-2-ylmethyl)-
isoquinolin-1-one10 and 2-(1-methyl-2-nitroimidazol-5-yl-
methyl)isoquinolin-1-one.12 In these studies, the release of
the drugs was studied qualitatively by thin layer chroma-
tography (TLC) and, in one case, by high performance
liquid chromatography (HPLC)12 but release from this type
of prodrug has not, as yet, been studied by NMR. The
analogous nitrothiophenes 1c have not hitherto been
investigated. The redox potentials of 2-nitrofurans are
relatively high (E17¼2325 mV for a 5-nitrofuran-3-carb-
oxamide)5 for this application; those of 2-nitroimidazoles
are more appropriate for selective bioreduction in hypoxic
tumour tissue (E17¼2389 mV for 1-alkyl-2-nitroimida-
zoles).5 Nitrothiophenes are somewhat less electron-affinic,
with 5-nitro-2-(oxiranylmethyl)thiophene having E17¼
2481 mV.15 A series of 5-nitrothiophene-2-carboxamides
has shown unexpectedly powerful radiosensitising activity
towards hypoxic tumour cells in culture.15 Thus it was
hypothesised that nitrothienylmethyl prodrugs of general
Five series of nitrothienylmethyl prodrugs were designed
to test this hypothesis. These series included examples of
type 5a (Fig. 1), where the drug is linked through oxygen,
and examples of type 5b, where the linkage is through
nitrogen. The drugs chosen for linkage to nitrothiophene
were aspirin (used in the treatment of rheumatoid arthritis16
and in the prophylaxis of cardiovascular disease), predni-
solone (rheumatoid arthritis17), isoquinolin-1-ones (inhibi-
tors of poly(ADP-ribose)polymerase (PARP),18 with
applications in cancer,19 inflammation20 and hypoxia-
reperfusion injury21) and nifedipine (a Ca2þ-channel
antagonist22). Clearly, the point of attachment of the
nitrothienylmethyl unit to aspirin has to be through the
oxygen of the carboxylic acid, making the ester 8 (Scheme
2) the synthetic target. Prednisolone 11 also carries only
oxygen-based functionality to which the heterocyclic trigger
could be attached. However, preliminary synthetic studies
showed that the primary alcohol of 11 and its alkoxide did
not react with halomethylthiophenes. Thus a linker had to be
inserted between the steroid and the trigger. Prednisolone
hemisuccinate, the monoester of 11 at the primary alcohol
with butanedioic acid, is a known prodrug of 11 in its own
right,23 being cleaved efficiently to 11 in vivo by esterases.
Thus the butanedioate diester 12 (Scheme 3) was designed
as the reductively triggered prodrug for prednisolone; as
with 8, the group to be expelled after reductive triggering is
a carboxylate.
The inhibitory activity of (5-substituted) isoquinolin-1-ones
depends critically on the arylamide motif in which the N–H
is held syn to the carbonyl oxygen. Masking of this
pharmacophore can be achieved either by alkylation at
oxygen (giving 1-alkoxyisoquinolines) or at nitrogen
(giving 2-alkyisoquinolin-1-ones). Thus both 1-(5-nitrothi-
enylmethyl)isoquinolines (e.g. 13–15, Scheme 4) and 2-(5-
nitrothienylmethyl)isoquinolin-1-ones (e.g. 19, Scheme 4)
were sought as potential prodrugs. Finally, attachment of the
nitrothienylmethyl unit to the heterocyclic nitrogen of the
Scheme 2. Synthesis of 8, the nitrothienylmethyl ester of aspirin. Reagents:
(i) Et3N, CH2Cl2.
Scheme 3. Synthesis of 12, the nitrothienylmethyl prodrug of prednisolone and prednisolone hemisuccinate. Reagents and conditions: (i) succinic anhydride,
DMAP, pyridine, 508C, 5 h; (ii) C6F5OH, dicyclohexylcarbodiimide, EtOAc, 48C, 16 h; (iii) prednisolone, DMAP, DMF, 208C, 2 days.

S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
3439
Scheme 4. Structures of O-linked prodrug nitrothienylmethoxyisoquinolines 13–15 and of PARP inhibitors 16–18 and attempted synthesis of a corresponding
N-linked nitrothienylmethylisoquinolinone 19. Reagents and conditions: (i) various nitrating conditions; (ii) 20, LiN(SiMe3)2, NaI, THF, DMF, 208C, 2 days;
(iii) HNO3, CF3CO2H, 2108C!208C, 16 h; (iv) HNO3, Ac2O, AcOH, 2108C, 40 min.
1,4-dihydropyridine nifedipine in 25 (Scheme 5) was
predicted to mask its pharmacological activity.
was converted virtually quantitatively to its pentafluoro-
phenyl active ester 10; this ester reacted selectively with the
most sterically unencumbered (primary) alcohol of pred-
nisolone 11 to give the candidate prodrug 12.
Synthesis of the nitrothienylmethyl ester 8 of aspirin
(Scheme 2) was achieved in moderate yield, as expected,
by careful treatment of the corresponding acid chloride 6
with 5-nitrothiophene-2-methanol 7. For the other ester-
linked prodrug 12 (Scheme 3), initial unsuccessful attempts
were made to couple commercially available prednisolone
hemisuccinate with the nitrothienylmethanol 7. The syn-
thesis was achieved by joining the Trigger and Linker units
first, then attaching the drug. Treatment of alcohol 7 with
succinic anhydride under basic conditions afforded the
monoester 9 in good yield. The remaining carboxylic acid
Whereas deprotonation of isoquinolin-1-ones and treatment
with haloalkanes usually gives products of alkylation at
nitrogen,10,12,24 we have recently reported that application
of the Mitsunobu reaction gives either alkylation at nitrogen
or at oxygen,25 depending on the precise conditions and on
the nature of the reactants. Starting with isoquinolin-1-one
16 (a relatively weak inhibitor of PARP)18 and its 5-iodo-
and 5-bromo analogues 17 and 18 (more potent inhibitors of
the enzyme),18 treatment with 5-nitrothiophene-2-methanol
7 under Mitsunobu conditions gave25 the required O-linked
candidate prodrugs 13–15 (Scheme 4), respectively.
However, synthetic approaches to the N-linked analogue
19 were more challenging. Whereas the anion derived from
16 reacts with 5-nitrofuran-2-ylmethyl tosylate to give the
N-(2-nitrofuran-2-ylmethyl)isoquinolinone in modest yield,
this reaction failed with 2-chloromethyl-5-nitrothiophene.26
An alternative strategy was then investigated, in which the
thienylmethyl unit is attached first and is subsequently
nitrated. Deprotonation of isoquinolin-1-one 16 with
lithium bis(trimethylsilyl)amide and reaction of the anion
with 2-chloromethylthiophene 2027 in the presence of
catalytic sodium iodide gave 2-(thien-2-ylmethyl)isoquino-
linone 21, as reported previously.25 Nitration of 21 with
nitric acid in trifluoroacetic acid under usual conditions for
nitration of thiophenes gave the dinitro compound 22 in
reasonable yield as the only isolable product. Several other
nitrating systems gave the same product but in lower yields.
This outcome parallels the dinitration10 of 2-(furan-2-
ylmethyl)isoquinolin-1-one under all but the mildest
conditions. Rationalising that milder conditions may give
selective nitration at the thiophene and mindful of the
precedent10 that 2-(furan-2-ylmethyl)isoquinolin-1-one can
be selectively nitrated at the furan, 21 was treated briefly
with acetyl nitrate (formed in situ from nitric acid and acetic
anhydride) at 2108C. However, mononitration took place at
the isoquinolinone 4-position solely, giving 23; thus the
isoquinolinone is more nucleophilic than the thiophene and
Scheme 5. Attempted synthesis of N-(nitrothienylmethyl)nifedipine
25. Reagents and conditions: (i) 2-chloromethyl-5-nitrothiophene or
5-nitrothien-2-ylmethyl 4-methylbenzenesulfonate, various conditions;
(ii) LiN(SiMe3)2, PhCH2Cl, NaI, THF, DMF, 208C, 2 days; (iii)
LiN(SiMe3)2, 20, NaI, THF, DMF, 208C; (iv) various nitrating conditions.

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S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
Scheme 6. Outcomes of chemical model reductive release experiments. Reagents: (i) Zn, NH4Cl, MeOH; (ii) NaBH4, Pd/C, aq. PriOH, 408C; (iii) SnCl2,
MeOH; (iv) NaBH4, Pd/C, aq. PriOH, 208C.
the desired selective reaction is unlikely to be possible, in
contrast to that of the analogous furan.
alkynes) and this reagent has been used in aqueous
propan-2-ol for biomimetic reductive triggering of release
of nitroheterocyclic prodrugs.10,12 Reductive debromination
of 5-bromoisoquinolin-1-one 18 was also reported12 with
this reagent system. Tin(II) chloride causes reductively
triggered release from 5-nitrofuran-2-ylmethyl10 and
1-methyl-2-nitroimidazol-5-ylmethyl12 prodrugs. Similarly,
zinc powder/ammonium chloride in wet methanol reduces
2-nitroimidazoles to a mixture of the corresponding
2-amino- and 2-hydroxylamino-imidazoles.12
Scheme 5 shows the attempted synthetic approaches to the
nitrothienylmethyl derivative 25 of nifedipine 24. Nifedi-
pine 24 is a 1,4-dihydropyridine in which the ring nitrogen
is a two-fold ‘vinylogous amide’ and thus is of low
nucleophilicity. Indeed, N-alkylations of analogous 1,4-di-
hydropyridines have only been achieved either using
reactive Mannich reagents28 or oxiranes29 or, after depro-
tonation, using activated alkyl halides30,31 or simple
electrophiles.32,33 In this study, nifedipine 24 was deproto-
nated with lithium hexamethyldisilazide and the anion was
treated with a model electrophile, benzyl chloride; the
N-benzyl derivative 26 was obtained in 40% yield.
However, this alkylation could not be transferred to the
desired attachment of the nitrothienylmethyl unit directly
using 2-chloromethyl-5-nitrothiophene.26 Alkylation of the
nifedipine-derived anion was achieved with chloro-
methylthiophene 2027 in poor yield but attempted selective
nitration of the thienylmethyl-nifedipine at thiophene
gave only products of oxidative degradation of the
dihydropyridine.
Initially, the Zn/NH4Cl reduction system (Method A) was
used to study its effects on aspirin nitrothienylmethyl ester
8. HPLC analysis of the reaction mixture indicated rapid
(,1 min) conversion of 8 to 5-nitrothien-2-ylmethanol 7
and aspirin 6, indicating that hydrolytic cleavage of the ester
bonds had taken place, without reduction of the nitro group.
Treatment of the analogous prednisolone hemisuccinate
nitrothienylmethyl ester 12 with tin(II) chloride in methanol
(Method B) gave no reaction but gentle warming (408C)
again led to ester cleavage without reduction of the nitro
function, as shown by HPLC analysis. Interestingly,
the steroid product was prednisolone hemisuccinate 26
(Scheme 6), rather than prednisolone 11. Thus, although
both substrates 8 and 12 are diesters, only the nitro-
thienylmethyl ester was cleaved and without prior reduction
of the nitro group. The mechanistic origin of this selectivity
is unclear. It is interesting to note that Everett et al.36
achieved only a very small yield of aspirin 6 upon radiolytic
reduction of its prodrug ester with 1-methyl-2-nitroimida-
zole-5-methanol in aqueous buffer.
We have previously reported three different chemical
reductant systems to mimic bioreduction of heterocyclic
nitro groups in hypoxic tissue.10–12 In each of these, the
conditions were designed not to permit hydrogenolysis of
the ‘benzylic’ CH2–O or CH2–N bonds, which would not
be biomimetic for the nitroreductases and the cytochrome
P450 reductase enzymes. Sodium borohydride in aqueous
methanol has long been used as a selective reductant for
nitro groups34 (although recent questions35 have been raised
about its complete selectivity, in that it also reduces
The nitrothienylmethyl prodrugs 13–15 of the isoquinolin-
1-ones 16–18 were too insoluble in methanol for these

S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
3441
Figure 2. 1H NMR spectra (400 MHz) of 5-halo-1-(5-nitrothien-2-ylmethoxy)isoquinolines 14 and 15 in CD3OD before and after treatment with NaBH4/Pd/C.
Reduction of the nitro group causes expulsion of the 5-haloisoquinolinones 17 and 18, respectively, while the aminothiophenes residues form aliphatic
co-products. The major peaks in the spectra after treatment correspond to those of pure 17 and 18 in the same solvent.
reductive systems (A and B) to be investigated. Addition of
chloroform to the tin(II) chloride/methanol reaction mixture
served to dissolve the substrates but no reductively-
triggered release was observed. Thus the method previously
used10 for reductively triggered release of isoquinolin-1-one
from 2-(5-nitrofuran-2-ylmethyl)isoquinolin-1-one was
adapted for this study. Each of the nitrothienylmethyl
prodrugs 13–15 was treated with sodium borohydride in the
presence of palladium on carbon in aqueous isopropanol.
TLC analysis indicated the disappearance of 13–15 and the
presence of the isoquinolin-1-ones 16–18, respectively,
after 30 min (Scheme 6). Proton NMR analysis of the
reaction mixtures reconstituted in tetradeuteriomethanol
was carried out for each sample after 16 h reaction. Each
spectrum showed complex peaks in the aliphatic region but
the region d 6.0–9.0 was clear. In each case, the spec0 tra
showed complete disappearance of the thiophene 3 ,40-
protons. Aminothiophenes, the expected initial products, are
unstable when lacking electron-withdrawing substituents
and fragment by ring-opening to aliphatic materials. The
sole peaks observable in the aromatic region corresponded
to those of the released isoquinolin-1-ones. The part spectra
for the studies on 14 and 15 are shown in Figure 2. In each
case, marked differences were observed in the chemical
shifts of the isoquinoline 3-H and 4-H before and after the
treatment with NaBH4/Pd, whereas those for the benzene
ring protons at positions (5), 6, 7 and 8 were less affected.
For example, in the case of the 5-iodo compound 14, the 3-H
signal moves upfield by 0.75 ppm upon reductively
triggered release, the 4-H signal moves upfield by
0.81 ppm, the 6-H signal moves upfield by 0.11 ppm, the
7-H signal moves upfield by 0.13 ppm and the 8-H signal
moves downfield by 0.02 ppm; the greater effect in the
heterocyclic ring probably reflects the change from the
‘alkoxypyridine’ structure to the carbonyl ‘pyridinone’
tautomer.
3. Conclusions
These release experiments indicate that the nitrothienyl-
methyl esters are not good potential prodrugs, since they are
prone to cleavage of the ester link between masking group
and drug, without reductive triggering. In contrast, the
1-(5-nitrothien-2-ylmethoxy)isoquinolines were highly
effective potential prodrugs of the potent PARP-inhibitory
(5-substituted) isoquinolin-1-ones, in that the biologically
active agents are released rapidly upon reduction of the
nitrothiophenes. This expulsion of the isoquinolinone
leaving group is irreversible, since the residual aminothio-
phene is chemically unstable and degrades to ring-opened
fragments. The isomeric 2-(5-nitrothien-2-ylmethyl)iso-
quinolin-1-ones, in which the PARP-inhibitory pharmaco-
phore (the trans arylamide) is also effectively masked, were
not synthetically accessible and thus could not be compared
for their efficiency of release of the 2-unsubstituted
isoquinolin-1-ones. Such a difference in efficiency of release
may exist,37 since the precise nature of the leaving group is
different (oxyanion vs nitrogen anion). Another potential
prodrug, the nifedipine derivative 25 in which the leaving
group would also be a nitrogen, was similarly synthetically
inaccessible. This comparison of leaving groups in reduc-
tively activated prodrugs will require the synthesis of
analogous isoquinolines carrying different redox-sensitive
Triggers.

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S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
4. Experimental section
hexylcarbodiimide (170 mg, 850 mmol) in EtOAc (11 mL)
for 5 h and was allowed to stand at 48C for 16 h. Filtration
and evaporation gave 10 (350 mg, 98%) as a pale buff oil:
IR nmax 1770, 1730, 1470 cm21; NMR dH 2.84 (2H, t, J¼
6.2 Hz, CH2CO), 3.04 (2H, t, J¼6.2 Hz, COCH2), 5.29 (2H,
s, CH2O), 7.04 (1H, d, J¼4.2 Hz, thiophene 3-H), 7.81 (1H,
d, J¼4.2 Hz, thiophene 4-H); dC 28.2, 28.7, 60.8, 126.9,
128.1, 145.3, 168.1, 170.8; MS m/z 426.0078 (MþH)
(C15H9F5NO6S requires 426.0070).
4.1. General
1H and 13C NMR spectra for characterisation of synthetic
compounds were recorded using CDCl3 as solvent. Melting
points were determined using a Reichert-Jung Thermo
Galen Koffler block and are uncorrected. Infra-red spectra
were recorded as potassium bromide discs, except where
noted. Mass spectra were obtained in the FAB positive
ionisation mode, except where noted. Experiments were
conducted at room temperature, unless otherwise stated.
Solutions in organic solvents were dried with MgSO4.
Solvents were evaporated under reduced pressure. The brine
was saturated. The stationary phase for flash column
chromatography was silica gel. HPLC was performed
using a semi-preparative Kromasil 10C18 column, a Jasco
PU-986 preparative pump and Jasco UV-975 detector.
MeOH was used as the eluant, with a flow rate of
5 mL min21 the injection volume was 0.020 mL. 1-(5-Nitro-
thien-2-ylmethoxy)isoquinoline 13, 5-iodo-1-(5-nitrothien-
2-ylmethoxy)isoquinoline 14, 5-bromo-1-(5-nitrothien-2-
ylmethoxy)isoquinoline 15 and 2-(2-thienylmethyl)iso-
quinolin-1-one 21 were prepared as previously described
by us.25
4.1.4. (5-Nitrothien-2-yl)methyl prednisolone-21-yl butane-
dioate 12. Prednisolone 11 (180 mg, 520 mmol) was stirred
with 10 (220 mg, 520 mmol) and 4-dimethylaminopyridine
(5 mg) in dry DMF (10 mL) for 2 days. The evaporation
residue, in CH2Cl2, was washed with aq. HCl (1 M) and
water and was dried. Evaporation yielded 12 (233 mg, 75%)
as a yellow–orange glass: IR nmax 3450, 1740, 1650, 1600,
1470 cm21; NMR dH 0.95 (3H, s, prednisolone 18-H3),
1.0–1.2 (5H, m, prednisolone 7,8,14-H5), 1.45 (3H, s,
prednisolone 19-H3), 1.45–1.95 (5H, m, prednisolone 9,12,
15-H5), 2.0–2.6 (4H, m, prednisolone 6,16-H4), 2.80 (4H,
m, COCH2CH2CO), 4.49 (1H, s, prednisolone 11-H), 4.90
(1H, d, J¼17.5 Hz, prednisolone 21-H2), 5.00 (1H, d, J¼
17.5 Hz, prednisolone 21-H), 5.28 (2H, s, OCH2), 6.00 (1H,
s, prednisolone 4-H), 6.27 (1H, d, J¼10.5 Hz, prednisolone
1-H), 7.04 (1H, d, J¼4.3 Hz, thiophene 3-H), 7.29 (1H, d,
J¼10.5 Hz, prednisolone 2-H), 7.81 (1H, d, J¼4.3 Hz,
thiophene 4-H); dC 17.0, 21.1, 23.9, 31.3, 32.0, 34.1, 34.6,
39.5, 47.0, 51.4, 55.4, 67.2, 68.3, 69.9, 89.5, 122.2, 125.0,
148.0, 157.0, 171.0, 172.0, 186.1, 206.1; MS m/z 602.2084
(MþH) (C30H36NO10S requires 602.2059).
4.1.1. 5-Nitrothien-2-ylmethyl 2-acetoxybenzoate 8.
2-Acetoxybenzoyl chloride 6 (620 mg, 3.1 mmol) in
CH2Cl2 (5 mL) was added to Et3N (475 mg, 4.7 mmol)
and 7 (500 mg, 3.1 mmol) in CH2Cl2 (5 mL) at 08C. The
mixture was stirred at 208C for 1.5 h, then washed with aq.
HCl (3 M), aq. NaHCO3 and brine. Drying, evaporation and
chromatography (hexane/EtOAc 1:1) gave 8 (360 mg, 35%)
as a yellow oil: IR nmax 1750, 1715, 1600, 1500 cm21; NMR
dH 2.31 (3H, s, Me), 5.41 (2H, s, CH2), 7.07 (1H, d, J¼
3.9 Hz, thiophene 3-H), 7.12 (1H, dd, J¼8.2, 1.1 Hz, Ph 3-
H), 7.33 (1H, dt, J¼7.4, 1.1 Hz, Ph 5-H), 7.61 (1H, dt,
J¼7.4, 1.5 Hz, Ph 4-H), 7.83 (1H, d, J¼3.9 Hz, thiophene
4-H), 8.04 (1H, dd, J¼7.8, 1.5 Hz, Ph 6-H); dC 21.0,
60.8, 122.0, 123.8, 126.0, 126.7, 128.0, 131.6, 134.4,
145.4, 150.8, 163.4, 169.3; MS m/z 322.0385 (MþH)
(C14H12NO6S requires 322.0385); Found: C, 52.28; H,
3.42; N, 4.35. C14H11NO6S requires C, 52.33; H, 3.42; N,
4.36%.
4.1.5. 4-Nitro-2-[(5-nitro-2-thienyl)methyl]isoquinolin-
1-one 22. Conc. HNO3 (0.050 mL, 800 mmol) was added
to 21 (50 mg, 207 mmol) in CF3CO2H (1.0 mL) at 2108C
and the mixture was stirred at 208C for 16 h. The pH of the
mixture was adjusted to 5 with aq. NaOH (2 M) and the
mixture was extracted with EtOAc. The extract was washed
with water, aqueous NaHCO3 and brine. Drying, evapo-
ration and chromatography (EtOAc/hexane 1:1) afforded 22
(28 mg, 42%) as an off-white solid: mp 70–738C; IR nmax
1638, 1598, 1570, 1392 cm21; NMR dH 5.40 (2H, s, CH2),
7.18 (1H, d, J¼4.1 Hz, thiophene 3-H), 7.65 (1H, t, J¼
7.0 Hz, isoquinoline 7-H), 7.80 (1H, d, J¼4.1 Hz, thiophene
4-H), 7.89 (1H, t, J¼7.0 Hz, isoquinoline 6-H), 8.51 (1H, d,
J¼7.8 Hz, isoquinoline 5-H), 8.70 (2H, m, isoquinoline
3,8-H2); dC 48.6, 123.8, 127.3, 128.2, 128.8, 130.1,
134.6, 135.4, 143.9, 160.9; MS m/z 332.0340 (MþH)
(C14H10O5N3S requires 332.0341).
4.1.2. 4-(5-Nitrothien-2-ylmethoxy)-4-oxobutanoic acid
9. Compound 7 (1.00 g, 6.3 mmol) was stirred with succinic
anhydride (630 mg, 6.3 mmol) and 4-dimethylaminopyri-
dine (5 mg, 41 mmol) in pyridine (5 mL) for 8 h at 508C.
The evaporation residue, in EtOAc, was washed with water
and was dried. Evaporation and chromatography (EtOAc)
gave 9 (1.23 g, 76%) as a white solid: mp 93–958C; (Found:
C, 41.20; H, 3.45; N, 5.61%. C9H10NO6S requires: C, 41.69;
H, 3.47; N, 5.40%): IR nmax 1730, 1690, 1470 cm21; NMR
dH 2.70 (4H, m, CH2CH2), 5.27 (2H, s, CH2), 7.03 (1H, d,
J¼5.0 Hz, thiophene 3-H), 7.25 (1H, d, J¼5.0 Hz, thio-
phene 4-H); dC 173.9, 171.9, 146.3, 128.3, 126.7, 60.4,
28.9; MS m/z 260.0230 (MþH) (C9H10NO6S requires
260.0228).
4.1.6. 4-Nitro-2-(2-thienylmethyl)isoquinolin-1-one 23.
Compound 21 (50 mg, 207 mmol) was stirred with conc.
HNO3 (65 mL, 1.0 mmol) and Ac2O (94 mL, 1.0 mmol) in
AcOH (5 mL) at 108C for 40 min. Water was added and the
mixture was extracted with EtOAc. The extract was washed
with aq. Na2CO3 (10%). Drying, evaporation and chroma-
tography (EtOAc/hexane 1:1) yielded 23 (20 mg, 40%) as
an off-white solid: mp 75–778C; IR nmax 1670, 1630,
1508 cm21; NMR dH 5.43 (2H, s, CH2), 7.01 (1H, m,
thiophene 4-H), 7.22 (1H, d, J¼3.8 Hz, thiophene 3-H),
7.33 (1H, d, J¼4.6 Hz, thiophene 5-H), 7.64 (1H, t, J¼
7.2 Hz, isoquinoline 7-H), 7.85 (1H, t, J¼7.2 Hz, isoquino-
line 6-H), 8.53 (1H, d, J¼6.8 Hz, isoquinoline 5-H), 8.67
4.1.3. 5-Nitrothien-2-ylmethyl pentafluorophenyl butane-
dioate 10. Compound 9 (220 mg, 850 mmol) was stirred
with pentafluorophenol (150 mg, 850 mmol) and dicyclo-

S. Ferrer et al. / Tetrahedron 59 (2003) 3437–3444
3443
(2H, m, isoquinoline 3,8-H2); dC 47.3, 123.5, 124.1, 125.7,
References
126.4, 127.0, 128.3, 128.6, 128.8, 134.1, 135.8, 136.0,
160.8; MS m/z 287.0488 (MþH) (C14H11O3N2S requires
287.0490).
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1
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Acknowledgements
We thank Mr R. R. Hartell and Mr D. Wood (University of
Bath) for the NMR spectra and Mr C. Cryer (University of
Bath) for the mass spectra. We are grateful to the Arthritis
Research Campaign for part financial support. S. F. held a
studentship from the Department of Medical Sciences,
University of Bath.
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